Conformational Dynamics in Glutathione S-Transferase

谷胱甘肽 S-转移酶的构象动力学

• 批准号: 6436574
• 负责人: WILLIAM M ATKINS
• 金额: $ 23.8万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-10 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6436574
• 关键词: Escherichia coli; X ray crystallography; acidity /alkalinity; biochemical evolution; crystallization; detoxification; directed evolution; enzyme model; enzyme substrate; fluorescence spectrometry; glutathione transferase; intermolecular interaction; isozymes; model design /development; molecular dynamics; molecular site; natural selections; nuclear magnetic resonance spectroscopy; physical model; protein purification; protein structure function; site directed mutagenesis; statistics /biometry; structural biology; thermodynamics

DESCRIPTION (provided by applicant): The glutathione S-transferases (GSTs) are a family of detoxification enzymes that metabolize environmental xenobiotics and drugs, including anti-cancer agents, by conjugating them to the tripeptide glutathione (GSH). GSTs also modulate oxidative stress by metabolizing lipid hydroperoxides and lipid hydroxy-enals. GSTs are likely to play a role in sensitivity to atherosclerosis, cataracts, and neurodegenerative diseases. As a canonical family of structurally related proteins, the GSTs provide a model for understanding the evolution of substrate diversity, which apparently correlates with the evolution of protein dynamics in some GSTs. The GSTA1-1 isoform has two unusual features that may uniquely contribute to its catalytic diversity as a detoxification enzyme. One feature is a catalytic Tyr with an unusually low pKa, which, possibly, provides electrostatic forces and increases solvation of the active site. The ionization state of this Tyr does not change during chemical steps of the catalytic cycle, and the function of the unusual ionization properties remains unknown. The second feature is a dynamic C-terminal helix, which undergoes ligand-dependent redistribution between 'open' and 'closed' conformations. Highly related, nearly structurally identical, GSTs possess C-terminal helices that are 'static' and remain either 'open' or 'closed.' This proposal explores the catalytic function of the Tyr ionization properties and of the C-terminal helix and, in particular, the hypothesis that the two features have co-evolved as an evolutionary bridge between primitive GSTs and highly evolved substrate specific isoforms. In order to understand the structure, function, and dynamics of the GST family, the specific aims of this proposal are: 1) to determine the stage of GSTA1-1 catalysis at which the C-terminus closes; 2) to determine the function of the unusual ionization properties of the active site Tyr; 3) to explore the molecular determinants of substrate diversity by directed evolution of GSTA1-1 and directed de-evolution of GSTA4-4. The techniques to be used include x-ray crystallography, NMR, and fluorescence of model ternary complexes, to monitor the C-terminal structure and dynamics. In order to determine whether the C-terminus must be closed in the transition state for the chemical step, linear free energy relationships will be exploited. The relationship, if any, between C-terminal dynamics and the ionization of the catalytic Tyr will be explored with stopped-flow kinetic approaches and steady state fluorescence with an engineered Trp reporter.

描述（由申请人提供）：谷胱甘肽S-转移酶（GST）是 一个代谢环境异生物学的排毒酶家族 和包括抗癌剂在内的药物通过将它们连接到三肽 谷胱甘肽（GSH）。 GST还通过代谢脂质来调节氧化应激 氢过氧化物和脂质羟基烯。 GST可能在 对动脉粥样硬化，白内障和神经退行性疾病的敏感性。作为 典型的结构相关蛋白质家族，GST提供了一个模型 了解底物多样性的演变，显然与 随着某些GST中蛋白质动力学的演变。 GSTA1-1同工型具有 两个不寻常的特征可能会唯一促进其催化多样性 解毒酶。一项功能是催化Tyr，异常低 PKA可能提供静电力并增加溶剂化 活跃的站点。在此期间，该Tyr的电离状态不会改变 催化循环的化学步骤和不寻常的功能 电离特性仍然未知。第二个功能是动态 C末端螺旋，它依赖配体的重新分布 “开放”和“封闭”构象。高度相关，几乎结构上 相同的GST具有“静态”的C末端螺旋，并且保持 “打开”或“关闭”。该提案探讨了Tyr的催化功能 电离特性和C末端螺旋，尤其是 假设这两个特征已作为进化桥共同发展 在原始GST和高度进化的底物特异性同工型之间。为了 了解商品及服务税家族的结构，功能和动态， 该提案的具体目的是：1）确定GSTA1-1的阶段 C末端关闭的催化； 2）确定 主动部位Tyr的异常电离特性； 3）探索 通过GSTA1-1的定向演化，底物多样性的分子决定因素 并指示GSTA4-4的脱位。要使用的技术包括X射线 晶体学，NMR和模型三元配合物的荧光，以监测 C末端结构和动力学。为了确定是否 C末端必须在化学步骤的过渡状态下关闭，线性 自由能力关系将被利用。之间的关系（如果有的话） 将探索C末端动力学和催化Tyr的电离 使用停止流动动力学方法和稳态荧光 设计的TRP记者。